Protein dynamics under force My laboratory is focused on understanding protein dynamics under force, of common occurrence in biology. Over the last 20 years of NIH funding, we have pioneered the development of protein engineering and force spectroscopy techniques that we have used to study muscle proteins, proteins and polysaccharides of the extracellular matrix, and since 2015, a new focus on studying proteins involved in bacterial adhesion. This MIRA proposal will unify our current studies of protein mechanics under a single funding mechanism. The most important discovery of force-spectroscopy over the past 20 years is that proteins do mechanical work when they fold against an opposing mechanical force. For example, the amount of mechanical work done by a folding titin Ig domain can be 2-3 times larger than that of the chemically powered motor. Given that titin is the third filament of muscle, determining the role played by titin folding in the force generated by a contracting muscle is a scientific question of fundamental significance. Many key questions need to be answered before concluding that titin folding is a significant contributor to the mechanical work done by a contracting muscle. This MIRA proposal will give us the flexibility to attempt to answer them. For example, a key prediction of our model is that activation of myosin II motors by Ca++ leads to a drop in the force experienced by titin, triggering delivery of work by folding. This proposal focuses on further developing a set of novel tools that we have designed to directly answer such questions. The outcome of our efforts is likely to be a revolution in our understanding of the molecular mechanisms of muscle contraction. Since 2015, a parallel research track in my laboratory has been the study of Gram-positive pili, giant single polypeptide proteins composed of hundreds of Ig like repeats that resemble the overall design of titin. In sharp contrast to the inherent extensibility of titin, pili incorporate isopeptide bonds that prevent its mechanical unfolding and resist the large forces experienced by bacteria during colonization, biofilm formation, and infection. Since the discovery of the isopeptide bond in the shaft pilin of S. pyogenes in 2007, numerous new structures are being reported for other Gram-positive pili. Under a mechanical load however, the structure of the pilin proteins will rapidly change, altering its antigenic surfaces and exposing new ones. This present a novel opportunity, well adapted to force-spectroscopy techniques, for identifying peptides that can bind to and destabilize Gram-positive pili rendering them susceptible to proteolytic degradation. This MIRA proposal is aimed at developing rapid assays, based on force spectroscopy, for blocking isopeptide formation in bacterial shaft pilins, thereby using pilin mechanics to greatly expand the possibility for rationally designed antibiotics.